Identification and association of polymorphisms in the interleukin-13 gene with asthma and atopy in a Dutch population.

Asthma and atopy are related conditions that may share similar genetic susceptibility. Linkage studies have identified a region on chromosome 5q that contains biologic candidates for both asthma and atopy phenotypes, including several proinflammatory cytokines. Interleukin (IL)-13, one of the candidate genes in the region, is directly involved in the regulation of immunoglobulin E and has been associated with both asthma and atopy. We sought to identify new polymorphisms in the IL-13 gene, and evaluated the involvement of a subset of these variants in asthma and atopy in a case-control study using probands and spouses from a Dutch asthma family study. IL-13 was sequenced in 20 probands and 20 unaffected spouses, and 10 polymorphisms were identified, four novel and six previously reported. Three single nucleotide (nt) polymorphisms (SNPs) were detected in the 5'-promoter region, two in intron 1, and five in exon 4. Only one of the exon 4 SNPs resulted in an amino-acid change (Arg130Gln). We analyzed three SNPs in IL-13 in an extended group of 184 probands and their spouses: one in the promoter region (-1111), the Arg130Gln (nt position 4257), and a 3' untranslated region SNP (nt position 4738). The most significant associations were observed to asthma (P = 0.005), bronchial hyperresponsiveness (P = 0.003), and skin-test responsiveness (P = 0.03) with the -1111 promoter. These results provide evidence that variation in the IL-13 gene is involved in the pathogenesis of asthma and atopy. Further investigation is required to determine which specific alleles or combination of alleles contribute to these phenotypes, and the possible downstream effects of the resulting change in IL-13 levels or activity.

From symptomatic treatments to causative therapy?

The search for genes that predispose individuals to develop common chronic diseases such as asthma, diabetes and Alzheimer's promises to give insights into their molecular pathogenesis. This will lead to the development of therapies that modulate the pathology, rather than the physiology of these diseases. As academia and the pharmaceutical industry increasingly focus on this challenge, the genetic dissection of Alzheimer's is spearheading attempts to shift the therapeutic paradigm away from symptomatic to curative treatments.

Association of a promoter polymorphism of the CD14 gene and atopy.

Atopy is generally considered to be caused by interaction of genetic and environmental factors. Recently, an association of a C-to-T transition in the promoter region of the CD14 gene on chromosome 5q31.1 and atopic phenotypes was reported in a population study of school children in the United States. The aim of the present study was to investigate the association of the C allele of the CD14/-159 with phenotypes of atopy and asthma in an adult Dutch population in which linkage of total serum IgE and bronchial hyperresponsiveness to chromosome 5q31-33 is present. We studied 159 probands with asthma and 158 spouses as controls. Phenotypes for asthma (e.g., bronchial hyperresponsiveness, physician's diagnosis) and for atopy (e.g., total serum IgE level, intracutaneous skin test, allergic rhinitis) were studied. In this population, homozygotes for the C allele had a higher number of positive skin tests and higher total serum IgE levels (in skin test-positive individuals) and subsequently, more self-reported allergic symptoms including rhinitis and hay fever, compared with subjects with CT and TT alleles. We conclude that the -159 C-to-T promoter polymorphism in the CD14 gene may result in expression of a more severe allergic phenotype.

Effect of sub-inhibitory concentration of chlorhexidine on lipid and sterol composition of Candida albicans.

The effect of a sub-inhibitory concentration of chlorhexidine on lipid and sterol composition of Candida albicans was investigated. The total lipid content of this yeast grown in the presence of chlorhexidine was reduced whilst the total sterol content was increased compared with control-grown cells. Lipids and sterol analyses of this yeast grown in the presence and absence of chlorhexidine are presented. Chlorhexidine-grown yeast had a higher level of phosphatidylethanolamine, phosphatidylcholine and monogalactosyldiacylglycerol. Lower proportions of phosphatidylinositol plus phosphatidylserine, phosphatidic acid and cardiolipin were found in C. albicans grown in the presence of the drug when compared with control-grown yeast. The major fatty acids in control-grown cells were C16 and C18. Drug grown-cells had higher proportions of palmitic acid (16:0) and stearic acid (18:0), but lower proportions of palmitoleic acid (16:1) and oleic acid (18:1). Chlorhexidine also decreased the unsaturated-to-saturated fatty acid ratio, while the C16/C18 ratios increased compared to control-grown cells. Differences in the fatty acid composition of major phospholipids and neutral lipids between drug and control-grown yeast were also detected. Sterol analysis of control-grown cells showed that the major sterol present was ergosterol (55.4% wt). A significant increase in ergosterol and obtusifoliol was observed in chlorhexidine-treated cells and a significant decrease in squalene and lanosterol. Our results suggested that chlorhexidine affected the lipid and sterol composition of C. albicans.

Identification of evolutionary conserved regulatory sequences in the 5' untranscribed region of the neural-specific ubiquitin C-terminal hydrolase (PGP9.5) gene.

The structure at the 5' end of the gene encoding neural-specific protein gene product 9.5 (PGP9.5) has been compared between two evolutionary distant species: the human and Monodelphis domestica. In contrast to the highly conserved coding sequences of the gene, only a 48% identity was found across a 1-kb stretch of 5' untranslated and untranscribed DNA. Promoter function studies performed on the human sequence identified a 233-bp CpG-rich minimal promoter. Truncation mutagenesis revealed the presence of essential positive cis-acting regulatory sequences within the region -182 to -123 relative to the transcription initiation site. Sequence alignment analysis of the human and Monodelphis promoter sequences showed 76% identity in this 59-bp region of the gene. A perfectly conserved 12-bp sequence (PSN) located within this region acts as a non-cell-specific activator of transcription in a heterologous reporter gene (pBLCAT2). PGP9.5 gene expression can be readily detected in human neuroblastoma cell lines but is absent in nonneuronal cell lines such as HeLa. Studies on the cell type specificity of the human PGP9.5 promoter demonstrated that in contrast to the endogenous gene, the promoter is active in HeLa cells. However, the promoter displays higher levels of activity in human neuroblastoma cell lines. A conserved 16-bp sequence located at -356 (motif 5) was able to reduce the activity of a heterologous minimal promoter specifically in HeLa cells. In conclusion, we have shown that expression of the PGP9.5 gene is regulated by evolutionary conserved positive and negative cis-acting sequences located in the untranscribed region of the gene.

Induction of 18A2/mts1 gene expression and its effects on metastasis and cell cycle control.

The metastasis associated 18A2/mtsI gene was inserted into the mammalian expression vector pMAMneo placing it under the control of the dexamethasone-inducible MMTV promoter. The construct was transfected into dexamethasone receptor negative F1 and receptor positive F10 cells of the B16 murine melanoma. The transferred gene was switched on in two transfectant clones of F10, by exposure to 10(-6) M dexamethasone, but not in clones of the receptor negative F1 line. One of the F10 transfectant clones (F10-192/10) was characterized further. A 13.5-fold increase in 18A2/mts1 transcripts was found in this clone upon exposure to dexamethasone. There was also a seven-fold increase in lung colonization in an experimental metastasis assay, together with increased expression of depolymerized tubulin and enhanced detection of p53 protein. The number of cells in the S phase increased by 2.5-fold following dexamethasone treatment of the clone. These data suggest a direct involvement of the 18A2/mts1 gene in lung colonization by the tumor cells. The 18A2/mts1 protein promotes tubulin depolymerization, sequesters the p53 phosphoprotein, and induces the cells to enter the S phase, but the relevance of these in the metastatic process remains to be elucidated.

Germline and somatic mosaicism in a female carrier of Duchenne muscular dystrophy.

The family of a male with Duchenne muscular dystrophy (DMD) and a deletion within the dystrophin gene has been studied. Polymerase chain reaction analysis of ectopic mRNA from peripheral blood T+B lymphocytes and the use of (CA)n repeat polymorphisms in and around the deleted region showed the proband's mother to be both a germline mosaic and a somatic mosaic for the deletion seen in her son. The mutation therefore occurred as a mitotic event early in embryogenesis.

Construction and features of lambda EMBL3cosW, a lambda replacement vector for detailed analysis of large regions of genomic DNA.

A phage lambda replacement vector, lambda EMBL3cosW, is described which expedites detailed analysis of large regions of chromosomal DNA. Two features of the vector aid this process. Firstly, the replaceable stuffer in lambda EMBL3cosW is flanked by SP6 and T7 promoters so that end-specific hybridisation probes can be rapidly generated from cloned inserts for identification of sequentially overlapping clones in genomic libraries. Secondly, because all the phage coding sequences in the vector (which are placed to the right of the replaceable stuffer) can be removed from cloned inserts by cleavage with NotI, restriction mapping of cloned inserts using partial digest strategies is greatly facilitated. Other features of the vector are: (1) strategically placed BamHI and XhoI sites for the cloning of genomic DNA partially digested with MboI or Sau3AI by two different methods; (2) SalI and SfiI sites for the isolation of intact cloned inserts; and (3) transcription terminators to insulate vector genes from transcriptional interference from cloned insert DNAs.

Mapping of 386 kb of genomic DNA in the human dystrophin-encoding gene (DYS) using an ordered phage lambda sublibrary of a YAC clone containing the DYS region.

An integrated restriction map for HindIII and EcoRI has been constructed for 386 kb of the human dystrophin-encoding gene by partial digest mapping of 35 overlapping lambda EMBL3cosW phage clones derived from a yeast artificial chromosome containing this region. Map construction was simplified in two ways. Firstly, the sequence arrangement of lambda EMBL3cosW is such that only map data from cloned inserts are generated using partial digests of lambda phage DNA asymmetrically labelled at the left cos end with a complementary 32P-labelled oligodeoxyribonucleotide. Secondly, the degree of partial digestion was standardised for each restriction enzyme by using ultraviolet light-induced formation of thymine dimers in the recognition sequence to partially block the cleavage reaction. The map provides the basis for work on the analysis of chromosomal rearrangements in this region which give rise to Duchenne muscular dystrophy, and for studies of chromosome structure and function.

Generation of ordered phage sublibraries of YAC clones: construction of a 400-kb phage contig in the human dystrophin gene.

A phage contig of 400 kb that extends from the brain-specific promoter at the 5'-end of the human dystrophin gene, through the muscle-specific promoter over 100 kb further downstream, and across most of intron 1 has been assembled. To achieve this, a yeast artificial chromosome (YAC) subcloning approach was used. Total DNA from a yeast strain containing a 400-kb YAC from the dystrophin gene was cloned using a lambda phage vector containing RNA polymerase promoters flanking the cloning sites. Phage containing human DNA inserts were then ordered into an overlapping set by hybridization of end-specific RNA probes from individual clones back to plaque lifts of gridded phage subclones. The clones generated will be useful as reagents for detailed structural and functional analyses of this region of the dystrophin gene.

Multiple transformation of Saccharomyces cerevisiae by protoplast fusion.

A technique for the multiple transformation of yeast by protoplast fusion is described. This involved the PEG-induced fusion of protoplasts from cells which had been treated with chromosome-fragmenting agents (in this case cupferron and hydroxylamine) with protoplasts of triply auxotrophic cells. The recovery of transformants was increased significantly if one of the amino acid requirements of the recipient strain was included in the selection medium. Transformants isolated on supplemented media remained auxotrophic for that requirement. Prototrophic, uninucleate transformants had a DNA content and cellular volume similar to that of the parental strains. Possible mechanisms of gene transfer are discussed. This technique offers the possibility of transferring desirable characteristics from one yeast strain to another without altering the ploidy level of the recipient strain.

Isolation and characterization of respiration-deficient mutants from the pathogenic yeast Candida albicans.

The isolation of several respiration deficient mutants of the pathogenic yeast Candida albicans is described. These show greatly reduced respiration rates, loss of cytochromes aa3 and b, and reduced growth rates. All of the mutants had lost the ability to assimilate a wide range of carbon sources. Ultrastructural studies showed reduced development of mitochondrial cristae in the mutants. The mutants can be divided into three classes depending on their respiration responses to the addition of cyanide.

Enhanced intraspecific protoplast fusion in yeast.

Intraspecific hybrid production from the polyethylene glycol induced fusion of yeast protoplasts was greatly increased when calcium propionate was included as the source of the requisite Ca2+. The use of calcium propionate, as opposed to the more commonly employed calcium chloride, resulted in substantially greater yields of hybrids from intraspecific fusions of protoplasts of Saccharomyces cerevisiae and Candida albicans. It is postulated that the ability of calcium propionate to enhance the fusion frequency is due to the anion binding to the etheric oxygen of PEG and potentiating the fusogenicity of the polymer.

The effect of mycophenolic acid on the cell cycle of Candida albicans.

Mycophenolic acid inhibited the growth of Candida albicans. Cultures exposed to a concentration of 8.4 micrograms ml-1 mycophenolic acid were found to exhibit cell cycle arrest with two or more buds. Nuclear staining revealed that these were nucleate implying a possible defect in cytokinesis. The results are discussed in relation to the possible mode of action of mycophenolic acid.

Antimycotic effects of octenidine and pirtenidine.

The effects of octenidine and pirtenidine on yeasts (in particular Candida albicans) have been studied. MIC and MCC values have been established as well as the inhibitory effects on growth, budding and germ tube formation. The drugs were shown to cause extensive leakage of cytoplasmic contents from the cells which was correlated with morphological and ultrastructural changes in the yeast.